Preliminary Study Of Tissue Distribution, Developmental Expression And Its Effect Of GK Gene In Schizothorax Prenanti

Glucokinase (GK) is a rate-limiting enzyme in glycolysis. To better understand of the GK molecular characteristics and regulation in Schizothorcx prenanti, we employed RACE to obtain the full-length cDNA of GK gene. Subsequently, real-time quantitative PCR (qPCR) was used to detect the tissue distribution of GK in adult fish, postpartum brood stock of S. prenanti, and chronological expression of GK in embryos and larvas which were born from the same biological parents. Finally, the changes of GK expression in S. prenanti which were fed different initial diets, stimulated with different sugars were examined by qPCR.The full-length GK cDNA (GenBank number:KT895594.1) fragment of S. Prenanti was 2087bp, and contained a 63bp of 5'-UTR, a 593bp of 3'-UTR and a 1431bp of ORF. The 476 amino acid residues deduced GK protein includes a GK-specific domain region, ATP-binding sites and glucose-binding sites. The S. Prenanti GK sequence had higher homology with Cyprinus carpio (97.9%), Carassius auratus var. Pengze (97.3%) and Ctenopharyngodon idella (96.8%).Tissue distribution of GK gene was highly specificity. The highest level of GK expression was observed in hepatopancreas relevant to 19 tissues of adult S. prenanti, followed by heart, ovary, testis, and little expression was found in other tissues. The highest GK expression was also in hepatopancreas relevant to 16 tissues of postpartum brood stock (female was higher than male), but were extremely low in heart, ovary, testis and other tissues. Breeding process affected the GK expression in tissues.The temporal expressions of GK were distinct in 26 embryonic and 10 post-embryonic development stages from the same parental S. prenanti. The GK expressions of post-embryonic stages were higher than embryonic stages. GK had been expressing from unfertilized egg, the difference was not significant until 4-cell stage (82.2?·h), and reached highest level at the 16-cell stage (127.5?·h), then declined. The expression quantity was close to unfertilized egg at early blastula phase (225.3?·h) and to rise again. GK expressions had been declining after the early gastrula stage (684.7?·h), except for a small peak arose at heart primordia occur (1619.2?·h). The GK expression level was lower than unfertilized egg before hatch out (2154.7?·h).GK expression level was low in newly hatched larvae and significantly increased at the phase of circulatory (4034.5 ?·h). Until the body pigment appearance (4406.5 ?·h), it began to decrease. After that, expression value continually rose, and reached the top at the digestive tract getting through (6278.5?·h) when larvae had flat swam for food that led to energy consumption increasing, then GK expression quantity began to fall. The GK expression level was still higher than unfertilized egg at Scales formed. The high expression of GK was related to the formation of the vital organs and high energy consumption in the process of embryonic and post-embryonic development. This study will provide an essential groundwork to the role of GK in the S. prenanti's entire life cycle.The GK enzyme activity and GK expression had obvious changes when fed egg yolks, artemia cysts, micro-diets to S. prenanti. larva. The different diets did not affect enzyme activity and mRNA expression of GK at 3d, proved that the yolk sac reduced the influence of diets on GK enzyme activity and gene expression. GK expression quantity was increasing with the increase of the culturing time in artemia cysts and micro-diets groups during 3d to 30d, while basically remain unchanged in egg yolks group (except the 10d was slightly higher). GK expression quantity was positively correlated to the growth speed of larvae, and raised with the increasing of culturing time. There was not reflected the corresponding relationship between GK enzyme activity and GK gene.The S. prenanti's hepatopancreas GK enzyme activity and gene expression were not changed when fed glucose and soluble starch at 1h, then both of them would increase after 1h (at 24h recovery the level of Oh), and glucose replied faster. It demonstrated that the glycolysis of glucose was more efficient in fish within 24h. Glucose inhibited hepatopancreas PEPCK enzyme activity and gene expression, but not suppressing PEPCK expression in kidney, foregut and hindgut. This study will provide an essential groundwork to further elucidate the essence of glucose metabolism of S. prenanti.